This project will continue ongoing studies that explore two specific characteristics of developing red cells: First, the accumulation of large amounts of mRNAs coding for the globin subunits of hemoglobin; and, second, the selective accumulation in red cells of the "erythroid" isoform of band 4.1. Studies of globin messenger RNAs will focus on thalassemia syndromes arising from premature translation termination codons in the mRNA coding sequence. These mutations not only ablate translation, but also impair accumulation of the mRNAs. Impaired accumulation seems to arise from defective nuclear/cytoplasmic transport, or from mechanisms that destabilize these mRNAs in erythroblasts. Natural and genetically engineered mutant globin genes will be used to synthesize RNAs that will be microinjected into the nuclei of foreign host cells in order to determine whether transport of pre-mRNAs or mature mRNAs is disrupted by premature termination codons. We shall also assess transcription, nuclear/cytoplasmic partitioning, and turnover of RNAs in erythroblasts and erythroid cell lines transfected with thalassemic genes. Once the abnormal step in mRNA metabolism is localized, it will be explored by assessment of the interaction of the abnormal mRNAs with riboonucleoprotein particles by methods recently developed for the isolation of RNA splicing complexes. By identifying the defect in mRNA accumulation, we hope to gain insights about the factors needed for promoting abundant accumulation of globin mRNAs in vivo. The second major thrust of this project will focus on our recent discovery of a seemingly widespread nucleotide sequence motif in a "non-erythroid" band 4.1 cDNA cloned in our laboratory. The "non-erythroid" isoform lacks a 21 amino acid segment in the spectrin-actin binding domain that is present in "erythroid forms of band 4.1. The "erythroid" isoform may thus be structurally unique among band 4.1 proteins produced in various cell types. We propose to determine whether the tissue specific predominance of these isoforms arises from multiple independently regulated genes or from a single gene via alternative mRNA splicing pathways. We shall analyze the transcription, post-transcriptional processing, and translation of the unique erythroid and non-erythroid mRNAs that we have identified by sequence analysis, in order to assess the steps in gene expression that determine the biogenesis of band 4.1 isoforms in erythroid and non-erythroid cells.